Elaboration of the plant body pattern depends primarily on the proper regulation of cell division versus cell differentiation at the growth sites called meristems. In seed plants, apical growth is carried out by the apical meristems. Although structurally identical, shoot apical meristems differ ontogenetically. A primary shoot apical meristem originates during embryogenesis and becomes the apex of the primary shoot. Secondary shoot apical meristems develop later-on the sides of the primary shoot and form lateral shoots. In many seed plants, radial growth of the shoot is conferred by the cambium, a cylindrical meristematic layer in the shoot body. Growth of lateral “leafy” organs (i.e., leaves, petals, etc.) occurs from transient meristems formed on the flank of the apical meristem. Root growth occurs from analogous apical and cambial meristems. Presently, very little of the regulation and interaction of these different types of meristems is understood.
The commercial value of a cultivated plant is directly related to yield, i.e. to the size and number of the harvested plant part, which in turn is determined by the number of cell divisions in the corresponding plant tissues. Although a genetic approach to the study of plant development has provided important information on pattern formation and organ morphogenesis (see, for example, Riechmann et al., 1997 Biol. Chem. 378:1079-1101; Barton, 1998 Current Opin. Plant Biol. 1:3742; Christensen et al., 1998 Current Biol. 8:643-645; Hudson, 1999 Current Opin. Plant Biol. 2:56-60; Irish, 1999 Dev. Biol. 209:211-220; Scheres et al., 1999 Current Topics Dev. Biol. 45:207-247). Very little has been learned about how and what regulates plant cell division, and, therefore the overall size of a plant organ. Therefore, the isolation and manipulation of genes controlling organ size via regulatory effects on cell division will have a large impact on the productivity of virtually every commercial plant species.
Hermerly et al. (1995 EMBO J. 14:3925-3936) studied the effects on tobacco plant growth and development using a dominant negative mutation of an Arabidopsis thaliana Cdc2 kinase gene. Cdc2 kinase activity is required in all eukaryotic organisms to properly progress through the cell cycle. Hermerly et al. showed that expression of the Arabidopsis thaliana gene encoding the dominant negative Cdc2 protein in tobacco plants resulted in plants that were morphologically normal, but were smaller in size due to a reduction in the frequency of cell division. Thus, the regulation of plant cell division can be at least partially uncoupled from plant development. However, in normal plant growth and development, Cdc2 kinase activity must be activated by other regulatory proteins in order to instigate plant cell division.
A large number of plant mutants have been isolated that display a wide variety of abnormal morphological and growth phenotypes (see, for example, Lenhard et al., 1999 Current Opin. Plant Biol. 2:4450). However, it is difficult to visually identify which plant morphology phenotypes are due to mutations in the putative key controller genes that determine whether a plant cell will grow and divide verses other genes that specify the developmental fate of a cell. Furthermore, even when such a putative plant growth mutant has been identified, a great deal of effort is required to identify which DNA segment encodes the mutant gene product that functions to regulate plant cell division.
For example, Talbert et al. (1995 Development 121:2723-2735), reported Arabidopsis thaliana mutants defective in a gene named revoluta (RET), that appear to display an abnormal regulation of cell division in meristematic regions of mutant plants. More specifically, the REV gene is required to promote the growth of apical meristems, including paraclade meristems, floral meristems and the primary shoot apical meristem. Simultaneously, the REV gene has an opposing effect on the meristems of leaves, floral organs and stems. That is, in leaf, floral organ and stem tissues REV acts to limit cell division, thereby, reducing both the rate of plant growth and final size of the tissue. Loss of functional REV protein in leaf, floral organ and stem tissues leads to an increase in the number of cells and the size of these tissues. In contrast, loss of functional REV protein in apical meristem cells leads to a reduction in cell division and reduced organ size. Talbert et al., (1995, incorporated herein by reference) reports the detailed morphological changes observed in homozygous revoluta plants. The aberrant morphologies recorded for revoluta mutants strongly suggest that the REV gene product has a role in regulating the relative growth of apical and non-apical meristems in Arabidopsis. The revoluta mutations were used to map the REV gene to the generally distal, but unspecified, portion of Chromosome 5 in Arabidopsis. However, prior to the present invention the REV gene sequence and methods for using polynucleotides encoding the REV protein to modulate cell division in transgenic plant cells were unknown.
In principle, mutations in a plant growth regulator gene could also be identified based upon their sequence similarity at the DNA or protein level as compared to animal or fungal genes that are known to play an important role in initiating the cell cycle (such as cyclins) or otherwise regulating growth. For example, homeobox (HB) genes are well know in animals as encoding proteins that act as master control genes that specify the body plan and otherwise regulate development of higher organisms (Gehring et al. 1994, Annu. Rev. Biochem. 63:487-526). The HB genes of animals encode an approximately 60 amino acid protein motif called a homeodomain (HD) that is involved in DNA binding, and the proteins that contain an HD are transcription factors which act as regulators of the expression of target genes. HD regions are highly conserved between both plants and animal. Plant homeobox genes were first identified based upon the isolation of a maize mutant called knotted1 (kn1) that had a dominant mutation that altered leaf development (Vollbrecht et al. 1991, Nature 350:241-243). Genes encoding proteins homologous to the maize Knotted protein have been identified and cloned from a wide variety of plant species based upon their sequence homology (for a review see Chan et al., 1998 Biochim. et. Biophys. Acta 1442:1-19). Hybridization studies indicate that there may be about 35 to 70 different HD-containing genes in Arabidopsis (Schena et al., 1992 Proc. Natl. Acad. Sci. USA 89:3894-3898).
A large number of plant HD-containing genes have been isolated using degenerate oligonucleotides made from conserved HD sequences as hybridization probes or PCR primers to identify and isolate cDNA clones (Ruberti et al., 1991 EMBO J. 10:1787-1791; Schena et al, 1992; Mattsson et al, 1992 Plant Mol Biol. 18:1019-1022; Carabelli et al., 1993 Plant J. 4:469-479; Schena et al., 1994 Proc. Natl. Acad. Sci. USA 91:8393-8397; Soderman et al., 1994 Plant Mol Biol. 26, 145-154; Kawahara et al., 1995 Plant Molec. Biol. 27:155-164; Meissner et al., 1995 Planta 195:541-547; Moon et al., 1996 Mol. Cells 6:366-373; Moon et al., 1996 Mol. Cells 6:697-703; Gonzalez et al., 1997 Biochem. Biophys. Acta 1351:137-149; Meijer et al., 1997 Plant J. 11:263-276; Sessa et al., 1998 Plant Mol. Biol. 38:609-622; Aso et al., 1999 Mol. Biol. Evol. 16:544-552). Analysis of these HD-containing genes revealed the presence of an additional large class of HD-containing genes in plants, known as HD-Zip genes because the proteins encoded by these genes contain a leucine zipper in association with the homeodomain. This class of HD genes are unique to plants (Schena et al., 1992). Based upon amino acid sequence similarity the proteins encoded by the HD-Zip genes have been divided into four HD-Zip subfamilies based upon the degree of amino acid similarity within the HD and leucine zipper protein domains (Sessa et al., 1994 In Molecular-Genetic Analysis of Plant Development and Metabolism [Puigdomenech, P. and Coruzzi, G., eds] Berlin: Springer Verlag, pp 411-426; Meijer et al., 1997). However, similar to the Knotted class of plant HD genes, the HD-Zip genes are also thought to encode proteins that function to regulate plant development (Chan et al., 1998). The presence of both HD and leucine zipper domains in the HD-Zip protein suggests very strongly that these proteins form multimeric structures via the leucine zipper domains, and then bind to specific DNA sequences via the HD regions to transcriptionally regulate target gene expression (Chan et al., 1998). This inference has been experimentally documented by in vitro experiments for many of the HD-Zip proteins (Sessa et al., 1993; Aoyama et al., 1995 Plant Cell 7:1773-1785; Ganzalez et al., 1997 Biochim. Biophys. Acta 1351:137-149; Meijer et al., 1997; Palena et al., 1999 Biochem. J. 341:81-87; Sessa et al., 1999), which publications are incorporated herein by reference.
Antisense and ectopic expression experiments have been performed with some HD-Zip subfamily I, II, III and IV genes to access the phenotypic consequences of shutting off HD-Zip gene expression and over producing HD-Zip protein throughout a plant (Schena et al., 1993; Aoyama et al., 1995; Tornero et al., 1996; Meijer et al., 1997; Altamura et al., 1998). Additional evidence regarding HD-Zip function has been inferred from in situ hybridization and Northern blot hybridization experiments to determine the temporal pattern of HD-Zip gene expression through plant development as well as to locate which specific plant cells or tissues exhibit HD-Zip gene expression (See Table 1). However, as demonstrated by the information compiled in Table 1, there is no clear pattern as to what regulatory roles HD-Zip proteins play in plant growth and development either as a super family or at the subfamily level. Furthermore, there has been no recognition that a HD-Zip gene product is involved in the regulation of plant cell division.
TABLE 1HD-Zip Genes And Their Proposed FunctionsHD-ZipSubfamilyand GeneExpression PatternProposed FunctionReferenceSubfamily IAthb-1late plant developmentactivation of genes related toAoyama et al., 1995leaf developmentAthb-3root and stem cortex?Soderman et al., 1994Athb-5leaf, root and flower?Soderman et al., 1994Athb-6leaf, root and flower?Soderman et al., 1994Athb-7low level throughout plant,signal transduction pathway inSoderman et al., 1994,induced by abscisic acid andresponse to water deficit1996water deficitCHB1early embryogenesismaintenance of indeterminantKawahara et al., 1995cell fateCHB2early embryogenesis?Kawahara et al., 1995CHB3mature tissue?Kawahara et al., 1995CHB4hypocotyl?Kawahara et al., 1995CHB5hypocotyl and roots?Kawahara et al., 1995CHB6late embryogenesis, mature?Kawahara et al., 1995tissueHahb-1stem?Chan et al., 1994VAHOX-1phloem of adult plantsdifferentiation of cambium cellsTornero et al., 1996to phloem tissueSubfamily IIAthb-2vegetative and reproductiveinvolved in light perception andSchena et al., 1993;(HAT4)phases of plant, induced by far-related responses in regulationCarabelli et al, 1993;red-rich lightof development1996; Steindler et al.,1999;Athb-4vegetative and reproductiveinvolved in light perception andCarabelli et al, 1993phases of plant, induced by far-related responsesred-rich lightHahb-10stems and roots?Gonzalez et al., 1997Oshox 1embryos, shoots of seedlingsleaf developmental regulatorMeijer et al., 1997and leaves of mature plantsSubfamilyIIIAthb-8procambial cells of the embryo,regulation of vascularBaima et al., 1995;induced by auxinsdevelopmentAltamura et al., 1998;Sessa et al., 1998Athb-9mRNA slightly enriched in stem?Sessa et al., 1998compared to leaf, root andflowerAthb-14Strongly enriched in stem, root,?Sessa et al., 1998slightly enriched in flowercompared to leafcrhb1expressed only in gametophyte?Aso et al., 1999Subfamily IVAthb-10 (G1-2)trichome cells and non-hairpositive regulator of epidermalRerie et al., 1994; Diroot cellscell developmentCristina et al., 1996;Masucci et al., 1996ATML1Expressed in L1 layer of theRegulation of epidermal cell fateLu et al., 1996shoot apical meristemand pattern formationHahr1Expressed in dry seeds,Early plant development?Valle et al., 1997hypocotyls and roots
The results summarized in Table 1 show that the regulatory role of any one individual HD-Zip gene product can not be predicted based upon which HD-Zip subfamily a gene is placed. The HD-Zip subfamilies were determined by alignment and comparison of the amino acid sequences found in the HD and leucine zipper domains (See Aso et al. 1999, FIG. 2 for the most recent HD-Zip region alignments). Conservation of HD-Zip regulatory function can be expected in many cases to depend on the extent of amino acid sequence similarity found in conserved protein domains found outside of the HD-Zip regions. That is, HD-Zip gene products from different plant species that are functional homologues to each other (i.e., perform the same biological function) are expected to not only share conserved HD-Zip regions, but show more amino acid sequence similarity over the entire length of the protein compared to other HD-Zip proteins that perform different biological roles. Thus, it is not surprising that the data summarized in Table 1 shows that there is no consistent pattern as to the inferred biological functions for individual HD-Zip I, II, III and IV gene products. Nonetheless, there is still wide-spread speculation that the proteins of the HD-Zip super family play important roles in regulating plant development (See, for example, Chan et al. 1998).
Given the agronomic importance of plant growth, there is a strong need for transgene compositions containing gene sequences which when expressed in a transgenic plant allow the growth of the plant to be modulated. The compositions and methods of the present invention allow useful transgenic plants to be created wherein cell division is modulated due to expression of a REVOLUTA transgene. Compositions and methods, such as those provided by the present invention, allow for controlling (including increasing) plant size via the ability to control (e.g., increase) the number of cell divisions in specific plant tissues.
The inventive compositions and methods provide another way to meet the ever increasing need for food and plant fiber due to the continual increase in world population and the desire to improve the standard of living throughout the world. Despite the recent agricultural success in keeping food production abreast of population growth, there are over 800 million people in the world today who are chronically undernourished and 180 million children who are severely underweight for their age. 400 million women of childbearing age suffer from iron deficiency and the anemia it causes results in infant and maternal mortality. An extra 2 billion persons will have to be fed by the year 2020, and so many more that will be chronically undernourished. For example, in forest trees the cambium is responsible for girth growth. In tomato (and many other plants), the ovary walls are responsible, not only for mature fruit size but also for soluble solids content. In cereal crops, the endosperm contributes to seed size. In some cases, increased yield may be achieved by lengthening a fruit-bearing structure, such as maize where the ear is a modified stem whose length determines the number of kernels. Moreover, possible use of transgenic plants as a source of pharmaceuticals and industrial products may require control of organ specific growth modulation.
The potential of a designer growth-increasing or growth-decreasing technology in agriculture is very large. A yield increase as small as a few percent would be highly desirable in each crop. Conversely, in many fruit crops it is highly desirable to have seedless fruits. The present invention, in addition to being applicable to all existing plant varieties, could also change the way crops are bred. Plant breeding could concentrate on stress and pest resistance as well as nutritional and taste quality. The growth-conferring quality of the present invention could then be introduced in advanced elite lines to boost their yield potential.